SYNTHESIS OF A HOMOVANILLIC-ACID IMMUNOGEN THAT INCORPORATES AN ISOSTERIC GROUP DESIGNED TO GENERATE ANTIBODIES WITH IMPROVED SPECIFICITY

There is only one description of a homovanillic acid immunogen that wa s used successfully to generate polyclonal antibodies for use in immun oassays for this metabolite of dopamine [Gallacher and Ho (1992) Bioge nic Amines, 9, 99-114]. Synthesis of an immunogen for homovanillic aci d requires covalent conjugation of the acid to carrier protein and thi s necessarily involves alteration of the molecular structure of the ac id. We designed a new homovanillic acid immunogen in which the hapteni c determinants more closely resemble free homovanillic acid than did t he conjugated homovanillic acid in the previously described immunogen. In the new immunogen (as in the earlier immunogen) homovanillic acid is linked to carrier protein through the 4-position of the metabolic. In the new immunogen, however, the 4-hydroxyl is replaced by an amide NH group, that resembles the OH group it replaces. The NH group is iso steric with an OH group and, like an OH group, can take part in hydrog en bonding interaction. The new immunogen was synthesised from 3-metho xyphenylacetonitrile. This was nitrated, and the 4-nitro product was i solated. The nitrile was then hydrolysed to the carboxylic acid which was protected as the methyl ester. The nitro group was then reduced to the amine and acylated with succinic anhydride thereby introducing th e amide at the 4-position. The remaining carboxyl group from the succi nimide linking moiety was coupled to carrier protein and the methyl es ter protection was removed by hydrolysis to give the new homovanillic acid immunogen. When the new immunogen was used to generate antisera i n sheep the resulting antisera exhibited higher titres than the earlie r antisera and, more importantly, were more specific for homovanillic acid.